Chromosomal multidrug resistance (MDR) in bacteria is a serious clinical problem. Our studies have shown that Escherichia coli becomes resistant to a variety of antibiotics, organic solvents and superoxides when the activities of any of three paralogous, but differently regulated, transcriptional activators, MarA, SoxS and Rob, are increased. These activators bind a sequence called the marbox which lies upstream of the promoters of a set of about 40 chromosomal genes called the marA/soxS/rob regulon. tolC and acrAB are regulon genes that have critical functions in multiple antibiotic resistance since their protein products constitute the most important MDR pump in E. coli. The major goals of this project are to understand the regulation of these activators, the mechanisms whereby they activate the regulon promoters, and the mechanisms whereby the MDR is generated. Upregulation of the transcriptional activators, MarA, SoxS and Rob can be effected by treating the cells with certain chemicals. Phenolic compounds derepress the marRAB operon;superoxides activate SoxR which in turn activates SoxS;and bile salts and other compounds activate the Rob protein directly. Thus, the upregulation of these activators can indicate the presence of such substances in the environment or in the cell. Previously, we found that E. coli tolC mutants, which do not have the TolC outer membrane channel, have elevated levels of transcription of marRAB and soxS and have elevated activity of Rob protein. Since TolC is a vital component of eight known efflux pumps in E. coli and plays important roles in ridding bacteria of multiple antibiotics, bile salts, organic solvents and other xenobiotics, we concluded (1) that in the absence of TolC, intracellular metabolic waste products accumulate and trigger the upregulation of the activators and (2) that TolC is normally involved in the efflux of cellular metabolites and not merely of xenobiotics. We have now found a second mutation that increases the transcription of one of the genes of the marA/soxS/rob regulon by about 9-fold. In a strain carrying this mutation and a tolC mutation, the increase is about 25-fold. We are characterizing this mutant to see if it affects marA, soxS and rob in the same way that they are effected by tolC mutations. Metabolomic and microarray studies will also be undertaken with both the single and double mutants to determine their effects on export of toxic metabolites. We have also identified six TolC-dependent pumps that affect the ability of E. coli to form colonies on MacConkey-lactose agar (MLA). MLA has been used for over one century to detect coliform or enteric bacteria as a means of testing for fecal contamination of water and dairy sources. It has long been known that tolC mutants can not plate on MLA. We used knock-out mutations, singly and in combination, to test the contributions to plating on MLA of the eight pumps known to require TolC as their outer membrane duct. As previously found with regard to toxic metabolite export, acrB and emrB play the most significant roles but, under certain conditions, mdtF and acrD can also contribute to growth on MLA. When the activities of these four genes were eliminated by mutation, insertions upstream of acrEF that increase the activity of acrEF and mutations in hns, a gene whose product (H-NS) normally silences acrEF, were found to allow plating. We also found that overexpression of the ompR or rob genes, but not soxS or marA, could activate acrEF. The effect of ompR required a wild-type rob gene but the rob effect did not require a wild-type ompR gene. Thus, it is likely that overexpression of OmpR activates Rob which then activates acrEF. This provides important new information on the previously unsuspected activation of rob by OmpR and activation of acrEF by Rob. Similar subsequent experiments showed that in the absence of acrB, emrB, mdtF, acrD and acrEF, rare mutations which are dependent on a functional macB gene could also allow the cells to form colonies on MLA. These mutations are now being analyzed. Thus, ability of E. coli to plate on MLA can involve the activities of up to six different TolC-dependent efflux pumps.